Chickenpox is caused by acute infection with varicella-zoster virus (VZV). The virus spreads throughout the body and enters cells of the nervous system. Latent infection occurs and the virus establishes itself in dorsal root and cranial nerve ganglia. The latent virus subsequently can reactivate and present as zoster (shingles). Researchers and pharmaceutical companies have developed chickenpox vaccines but the side effect of shingles due to the live virus establishing a latent infection is still of concern. The ability of a live virus vaccine to enter and maintain a latent infection phase therefore can compromise the safety of live viral vaccines. Any change to the virus that decreases the probability of establishing or maintaining a latent infection can bring significant public health benefits.
Live vaccines are very popular despite the possibility of latent infection. For example, the live attenuated VZV vaccine based on the “Oka virus” (see, U.S. Pat. No. 3,985,615) prevents chickenpox but the virus used in this vaccine can enter a latent infection phase in vaccinated individuals and later cause zoster (Sharrar et al., Wise et al.). The Oka virus is attenuated. However the reason for this attenuation and its significance to the latency problem is unknown. Improved vaccines both for humans and for veterinary care, are needed that comprise altered viruses that present less risk of establishing or maintaining a latent infection and therefore less likely to reactivate.
Recombinant DNA technology can be used to alter viruses. For example, as shown in FIG. 1, the VZV genome is 124,884 bp in length (line 1) and contains unique long (UL), unique short (US), internal repeat (IR) and terminal repeat (TR) regions (line 2). Of the VZV genes, ORF63 and ORF70 encode the same ORF63 protein sequence. According to several reports, the ORF63 genes are active during latency of VZV (Mahalingham et al 1996; Lungu et al 1998; Debrus et al 1995; Cohrs et al 2000; Kennedy et al 2000; Kennedy et al 2001; Sadzot-Delvaux 1995). However, Sommer et al. reported viral “replication required at least one gene copy” and therefore deletion of the ORF63 gene copies would not be helpful for making a live vaccine (J. Virology 75: 17 p. 8224-8239 September 2001). The ORF63 protein is present in virions (Kinchington et al 1995) and is a phosphorylated protein in VZV infected cells (Ng et al 1994). ORF63 protein can be phosphorylated by the VZV ORF47 protein kinase (Kenyon et al 2001) and by casein kinases I and II (Bontems et al 2002; Stevenson et al 1996). However, the VZV ORF47 protein kinase is not required for VZV replication (Heineman et al 1995).
The ORF63 protein contains a nuclear localization signal (Stevenson et al 1996) that may be involved in regular functioning of this protein. ORF63 protein for example localizes to the nucleus in infected cells, and to a lesser extent to the cytoplasm (Debrus et al 1995). In contrast, during latent infection ORF63 protein is located in the cytoplasm (Mahalingham et al 1996); during reactivation the protein moves to the nucleus (Lungu et al 1998). Deletion of the nuclear localization signal or mutations of serine and threonine residues (important for phosphorylation of the protein) in the carboxy half of the protein to alanine residues results in increased localization of the protein to the cytoplasm.
Thus, the search for technical improvements to replication competent vaccines needs new paradigms for selecting genetic weak points and making intelligent changes that can alleviate the latent infection problem.